Cell Penetration Profiling for Biotherapeutics Biotechnology promises the ability to control biology and disease with laser-like precision. Hundreds of peptides, proteins and nucleic acids are being developed as diagnostics and therapies, but nearly all of these have poor cell penetration and unpredictable subcellular localization. Currently, there are no quantitative, high- throughput tools to measure how much of a biomolecule enters a cell and where it distributes within the cell. The imprecision and low throughput of current methods for measuring intracellular delivery is a major barrier for the development of biomolecule therapies. The Kritzer lab has devised a new method for quantitating cell penetration, called the ChloroAlkane Penetration Assay (CAPA). CAPA measures the degree to which a molecule penetrates the cytosol, independent of the molecule's target or biological function. Our published and unpublished data show that CAPA is quantitative, inexpensive and high-throughput, and that it exclusively measures penetration to single cellular compartment. Based on these results, we envision that CAPA could be used for comprehensive cell penetration profiling for a large variety of biomolecules and drug delivery systems. This will provide detailed data on dose dependence, time course, penetration to different subcellular compartments, and cell type specificity. It will also allow independent profiling of endocytic uptake, endosomal escape and nuclear import. We propose applying cell penetration profiling to uncover structure-activity relationships for cell penetration for several classes of bioactive peptides, cell-penetrant proteins, and antisense oligonucleotides. Finally, we use cell penetration profiling to understand and improve the delivery of Cas9-sgRNA complexes by lipid nanoparticles. Our understanding of how all these classes of biotherapeutics reach the cell interior is severely limited by current methods. Cell penetration profiling provides a rapid means of measuring endocytic uptake, endosomal escape, and nuclear import, allowing critical drug delivery problems to be directly assessed and avoided. Since intra-cellular delivery is the major hurdle for most biotherapeutics, cell penetration profiling has the potential to become a routine and valuable part of the development process for peptide, protein and nucleic acid therapies.